1. Field of the Invention
The present invention is directed to the isolation, purification and structural identification of the bioactive component of water extracts of Cat's Claw (Uncaria species). The bioactive component previously was identified in vitro as quinic acid lactone and other related quinic acid esters. The present invention now identifies the in vivo bioactive component as quinic acid and quinic acid salts, including quinic acid ammonium salts. The present invention also is directed to the pharmaceutical use of said bioactive component for enhancing the immune, anti-inflammatory, anti-aging, anti-tumor and DNA repair processes in warm blooded animals.
2. Discussion of the Related Art
Uncaria tomentosa, commonly known as Una de Gato or Cat's Claw, has been widely used historically as a natural remedy, and is currently present in a number of nutritional formulations to treat a large variety of health disorders. To applicant's knowledge, all of the commercial preparations of Cat's Claw except the water soluble extract (the “Pero extract”) disclosed in U.S. Pat. Nos. 6,039,949 and 6,238,675 B1 and allowed patent U.S. Ser. No. 09/824,508, issued as U.S. Pat. No. 6,361,805 B2 (the “Pero patents”) to Pero, and U.S. Pat. No. 6,797,286 to Bobrowski, are based on the oxindole alkaloid content thereof. This is due to Dr. Keplinger's (Austria) discovery, in the early 1960's, of the presence of oxindole alkaloids. (Keplinger, K., Laus, G., Wurm, M., Dierich, M. P., Teppner, H., “Uncaria tomentosa (Willd.) DC.-Ethnomedicinal use and new pharmacological, toxicological and botanical results,” J. Ethanopharmacology 64:23-34, 1999). The Pero extract, the preferred embodiment of which is commercially available under the names C-MED-100® and ACTIVAR AC-11™, is a novel Cat's Claw extract quite unlike any other commercial versions in that it contains only traces of alkaloids (<0.05%) (Sheng et al., “Treatment of chemotherapy-induced leucopenia in the rat model with aqueous extract from Uncaria tomentosa,” Pytomedicine 7(2): 137-143, 2000). Instead, the Pero extract contains a new class of active ingredients, carboxyl alkyl esters (CAEs), having demonstrated efficacy as described and protected in the Pero patents. C-MED-100® and ACTIVAR AC-11™ are the first products offered in the nutritional industry to support both auto-immune and DNA repair-enhancing functions, which are of critical importance in reducing the consequences of age-related disorders such as autoimmune, inflammatory and neoplastic diseases. References herein to C-MED-100® and/or ACTIVAR AC-11™ shall be understood to include the Pero extract, of which C-MED-100® and ACTIVAR AC-11™ are preferred embodiments.
The precise chemical identification of the Pero extract's active ingredients has not heretofore been achieved. However, the chemical and biological characteristics of those ingredients have been sufficiently completed to standardize the commercial manufacture of the Pero extract. (See, the Pero patents).
C-MED-100® and ACTIVAR AC-11™, which are the commercially available Pero extract, are formulated and based on the historical medicinal uses of Cat's Claw, of which an important step is exhaustive hot water extraction for approximately 18 hours at around 95° C. The extract is then ultrafiltrated to remove high molecular weight (>10,000 MW) toxic conjugates, and spray dried to contain 8-10% carboxy alkyl esters (CAEs) as active ingredients in vitro. CAEs were characterized as the only active ingredients of C-MED-100® in vitro as a result of their absorption (85%) onto charcoal. No biological activity was observed in the unabsorbed fraction. Using thin layer chromatography (TLC) as the purification tool, the active ingredients showed a UV absorption maximum at about 200 nm, and reacted with hydroxylamine and ferric chloride, thus characterizing them as esters (e.g. CAEs).
The inventor has subsequently determined that the active ingredients of C-MED-100® and ACTIVAR AC-11™ in vivo are quinic acid, as free acid, and its salts, including quinic acid ammonium salt. There are two physiological factors regarding the natural forms of quinic acid as the active ingredients of water extracts of Cat's Claw such as C-MED-100® or ACTIVAR AC-11™ which, in turn, might result in quite different biological responses when administered in vitro or in vivo. First, the acidity of the stomach, pH=1, has been shown to be strong enough to hydrolyze any quinic acid esters present in C-MED-100® to quinic acid. Second, the microflora of the digestive tract of mammals are well known to both synthesize and metabolically convert quinic acid to other analogs such as chlorogenic acid, ferrulic acid, shikimic acid, cinnamonic acid, and benzoic acid. (Seifter E., Rettura G., Reissman D., Kambosos D., Levevson S. M. 1971, “Nutritional response to feeding L-phenylacetic, skikimic and D-quinic acids in weanling rats,” J. Nutr. 101(6): 747-54; Gonthier M. P., et al. 2003, “Chlorogenic acid bioavailability largely depends on its metabolism by the gut microflora in rats,” J. Nutr. 133(6): 1853-63). These well known physiologic facts have raised the possibility that even though quinic acid esters are the bioactive ingredients in vitro, quinic acid esters in vivo could have been metabolized to quinic acid before being absorbed into circulation to mediate efficacious responses. The research disclosed herein confirms that this is the case.
Daily oral doses of C-MED-100® between 250-700 mg have proven efficacious in humans. These dosages have been shown to enhance anti-inflammatory, DNA repair, immuno and anti-tumor processes of warm blooded animals, including humans. (See, the Pero patents; Lamm, S., Sheng, Y., Pero, R. W., “Persistent response to pneumococcal vaccine in individuals supplemented with a novel water soluble extract of Uncaria tomentosa, C-Med-100,” Phytomed 8: 267-274, 2001; Sheng, Y., Li, L., Holmgren, K., Pero, R. W., “DNA repair enhancement of aqueous extracts of Uncaria Tomentosa in a human volunteer study,” Phytomed 8: 275-282, 2001; Sheng, Y., Bryngelsson, C., Pero, R. W., “Enhanced DNA repair, immune function and reduced toxicity of C-MED-100™, a novel aqueous extract from Uncaria tomentosa,” J. of Ethnopharmacology 69:115-126 (2000)).
The CAEs in C-MED-100® are shown to give profound nutritional support as a dietary supplement because the CAEs enhance both DNA repair and immune cell responses, which, in turn, are the critical physiological processes that regulate aging. (See, the Pero patents, Sheng, Y., Pero, R. W., Wagner, H., “Treatment of chemotherapy-induced leukopenia in a rat model with aqueous extract from Uncaria tomentosa,” Phytomedicine 7(2):137-143 (2000) and as cited above). Both of these processes involve regulating the nuclear transcription factor kappa beta (NF-κB). NF-κB is well known to control (i) the nuclear events that salvage cells from apoptotic cell death and (ii) pro-inflammatory cytokine production. (Beg, A. A. and Baltimore, D., “An essential role for NF-κB in preventing tumor necrosis factor alpha (TNF-α) induced cell death,” Science 274: 782-784, 1996; Wang, C-Y, Mayo, M. W., Baldwin, A. S., “TNF-α and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-κB,” Science 274: 784-787, 1996). Hence, this mechanism directly connects induction of apoptosis to programmed cell toxicity with inhibition of pro-inflammatory cytokine production and inflammation.
Apoptosis is an essential biochemical process in the body that regulates cells from division (replication) into differentiation and toward an increased functional capacity. Cells entering apoptosis will not only be stimulated to differentiate and increase functionality but will eventually die from this “programmed cell death”. Thus, induced apoptosis resulting from NF-κB inhibition by C-MED-100® would (i) effectively kill tumor cells, because they would be forced out of replication by apoptosis and into eventual death; and simultaneously (ii) increase immune cell responsiveness, because more immune competent cells would be forced to differentiate and would live longer because of the parallel enhancement of DNA repair.
NF-κB also sends signals to inflammatory cells instructing them to produce cytokines (growth factors, i.e., TNF-α and the interleukins). These signals, in turn, stimulate phagocytic cells to kill more invading infectious agents, which, at least in part, is accomplished by producing high levels of oxygen free radicals. Thus, inhibiting NF-κB has anti-inflammatory properties because it prevents over-reaction of the inflammatory process that can be harmful to normal body tissues. In addition, because pro-inflammatory cytokines are a major source of endogenous free radical production in humans, NF-κB inhibition is antimutagenic by reducing genetic damage that may accumulate over the years. As fewer radicals are produced, there is less damage to the DNA and less inhibition of natural repair. A result is that aging is curtailed.
It is now shown that quinic acid and its salts, including quinic acid ammonium salt, have an effect on NF-κB in vivo corresponding to the effect of CAEs in vitro.
The Pero extract, preferably C-MED-100® or ACTIVAR AC-11™, is thus an ultimate nutritional supplement for anti-aging remedies because it prevents free radical damage by NF-κB inhibition, induces differentiation and immune cell responsiveness by apoptosis, enhances DNA repair, and kills tumor cells, which in turn are the major factors related to aging. (Sheng, Y., Pero, R. W., Amiri, A. and Bryngelsson, C., “Induction of apoptosis and inhibition of proliferation and clonogenic growth of human leukemic cell lines treated with aqueous extracts of Uncaria Tomentosa,” Anticancer Research 18:3363-3368(1998); Sandoval-Chacon M., Thompson J. H., Zhang X. J., Liu X., Mannick E. E., Sadowicka H., Charbonet R. M., Clark D. A., Miller M. J., “Anti-inflammatory actions of cat's claw: the role of NF-kappa B,” Aliment Pharmacol. Ther. 12: 1279-1289, 1998; Sandoval M., Charbonnet R. M., Okuhama N. N., Roberts J., Krenova Z., Trentacosti A. M., Miller M. J., “Cat's claw inhibits TNF alpha production and scavenges free radicals: role in cytoprotection,” Free Radicals Biol. Med. 29(1): 71-78, 2000; Åkesson C., Lindgren H., Pero R. W., Leanderson T., Ivars F., “An extract of Uncaria Tomentosa inhibiting cell division and NF-κB activity without inducing cell death,” International Immunopharm 3:1889-1900 (2003)). It is beneficial to identify the active component thereof. By isolating and identifying the active component, it is possible to purify the component and enhance the pharmaceutical use and increase the efficacy thereof.
The present invention is directed to the isolation, purification and identification of the CAEs characterized as the active ingredients of the Pero extract in vitro, which CAEs are identified and structurally elucidated as quinic acid analogs. The present invention also is directed to the isolation, purification and identification of quinic acid and quinic acid salts, including quinic acid ammonium salt, as the active ingredients of the Pero extract in vivo. The present invention also is directed to the use of quinic acid and quinic acid salts, including quinic acid ammonium salt, in vivo to enhance immune competency, treat disorders associated with the immune system, inhibit the inflammatory response, treat disorders associated with the inflammatory response, enhance the DNA repair process, enhance the anti-tumor response, and treat disorders associated with the response to tumor formation and growth.